China has a heavy burden of hepatocellular carcinoma, which is a serious threat to people′s life and health. However, the available drugs for advanced hepatocellular carcinoma in the past are limited and the efficacy is not satisfactory. In recent years, immunotherapy has a significant effects in some tumors. The authors introduce the efficacy of restart immunotherapy on an advanced hepatocellular carcinoma patient undergoing interruption of treatment due to corona virus disease 2019, in order to provide references for the diagnosis and treatment of this kind of patients.

Objective To investigate the feasibility of glycogen synthase kinase (GSK) 3βinhibitor CHIR99021 induces human embryonic stem cells to differentiate into definitive endoderm cells. Methods Human embryonic stem cells H1 cells were cultured in feeder-free medium (Essential 8). The expression of human embryonic stem cell pluripotency markers Oct4, Nanog were detected by immunolfuorescence. Human embryonic stem cells were treated with CHIR99021 of different concentrations (0.33, 1.00, 3.00, 9.00, 27.00μmol/L) and cultured for 3 d respectively. The untreated cells were taken as control. The expression levels of pluripotency related genes OCT4, SOX2 and deifnitive endoderm related genes GATA4, SOX17 were detected by lfuorescent quantitative polymerase chain reaction (PCR). The experimental data were compared using one-way analysis of variance and LSD-t test. Results The expression of human embryonic stem cell pluripotency markers Oct4, Nanog could be detected by immunofluorescence. The human embryonic stem cells were observed in good growth when treated with CHIR99021 of different concentrations (0.33, 1.00, 3.00μmol/L) . The human embryonic stem cells were observed in poor growth or dead when treated with the higher concentrations of CHIR99021. The expression of pluripotency related gene OCT4 decreased after treated with CHIR99021 of 1.00, 3.00μmol/L concentration (LSD-t=-40.54,-59.12;P<0.05). The expression of SOX2 also decreased significantly (LSD-t=-20.46,-3.87;P<0.05). The expression of definitive endoderm related genes GATA4 increased after treated with CHIR99021 of 1.00, 3.00μmol/L concentration (LSD-t=137.21, 65.29;P<0.05). The expression of SOX17 also increased significantly (LSD-t=50.93, 6.56;P<0.05). Conclusions Human embryonic stem cells can maintain a good pluripotent state in the feeder-free culturing system. CHIR99021 can effectively induce the human embryonic stem cells to differentiate into deifnitive endoderm cells.

ObjectiveTo investigate the synergistic cytotoxic effects and the mechanism of oncolytic adenovirus SG611 combined with cisplatin (DDP) on hepatocellular carcinoma (HCC) HepG2 cells. MethodsHuman HCC HepG2 cells were infected by adenovirus vector SG611 carried with green fluorescent protein (GFP). The infection efficiency of SG611 on HepG2 cells were examined by flow cytometry. The synergistic cytotoxic effects of SG611 combined with DDP on HepG2 cells were evaluated by cell counting rit(cck)-8 assay and the cytotoxicity was assessed by crystal violet staining. The 4',6-diamidino-2-phenylindole (DAPI) staining was used to detect the apoptosis. The expression of protein E1A was examined by Western blot. The comparison of experimental data was conducted using one-way analysis of variance and LSD-t test.Results HCC HepG2 cells infected by SG611-EGFP were observed under lfuorescence microscope. The GFP positive cells increased apparently with the increasing multiplicity of infection (MOI). The infection efficiency of SG611 detected by flow cytometry was 0.18%, 6.36%, 50.60%, 73.20%, 86.80% and 90.50%, which was with dose dependent. With the combined use of SG611 (MOI =10) and 1.5 μg/ml DDP, the cell viability was (33.2±1.2)%, which was significantly lower than (88.8±8.9)% of single use of SG611 (LSD-t=-7.83,P<0.05). The cytotoxic effects on HCC HepG2 cells of combined use was signiifcantly higher than that of single use of SG611. The apoptosis rate of HCC HepG2 cells was (23.9±1.5)% for combined use, which was signiifcantly higher than (15.3±1.0), (12.4±1.1)% for single use of SG611 or DDP (LSD-t=10.56, 21.34;P<0.05). In addition, E1A expression in HCC HepG2 cells significantly increased when combined use.Conclusions Oncolytic adenovirus SG611 combined with DDP has synergistic cytotoxic effects on HCC HepG2 cells. The action mechanism of the synergistic cytotoxic effects may be that the chemosensitivity of DDP is enhanced by SG611 and the proliferation of SG611 is enhanced by DDP.